Structure of tubulin and its organization in microtubules:

Tubulin = α + β polypeptides. (α & β bind tightly to each other, don’t come apart; act as a single unit.) Fig 20-3 (5th Ed.)

(microtubule = 13 protofilaments aligned side by side, slightly staggered upward) Fig 18-3 (b) (6th Ed.) This figure shows the seam along the length of the microtubule:

Tubulin has a (+) end and a (-) end. Subunits come on and off at both ends, but at concentrations of tubulin exceeding the critical concentration, net addition of tubulin onto the (+) end is more rapid than at the (-) end. Fig 20-7 (5th Ed.)

Fig 18-9 (6th)

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Two well-known drugs thct alTect nnicrotubules ere Colchicine and Taxol

At high concentations, colchicine (and a synthetic dorivative, colcemide) causes depolymerization ofmicrotubules; at low concentrations, it "freezes" microtubules. When added at low concentrations togowing cells, cells accumulate in M phase, where they are blocked in metaphase. (After washingaway the oolohicines, cells resume mitosis.)

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Taxol has the opposite effect: it hyper-stabilizes microtubules, driving the equilibrium in the cell suchthat all tubulin in polymerized, and dynamic instability is lost. This is just albad for the events ofmitosis as MT depolymerization or "freezing" caused by colchicines. Taxol has proved to be a veryeffective anti-cancer drue.

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Our cells are shaped and supported by a cytoskeleton ofinterlocking protein filaments. A beautiful star of micro-tubules, the largest of these filaments, radiates outwardfrom the center of the cell to the cell surface. This “aster”of microtubules is the railway system of the cell. Manytypes of cargo are carried along these rails. The endoplas-mic reticulum is pulled by molecular motor proteins alongmicrotubules, spreading it evenly throughout the cell.Vesicles are delivered to their destinations along micro-tubules. And, when cells divide, the most valuable cargo ofthe cell is carried by microtubules. Paired copies of each ofthe chromosomes are attached to the ends of a doubledmicrotubule aster and carefully separated into the twodaughter cells.

The cytoskeleton, in contrast to our articulated skeleton ofbones, is a dynamic structure. It is continually constructed anddemolished according to the shifting needs of the cell. A typ-ical microtubule lasts for only 10 minutes before it is disas-sembled and the parts used to build a new one. Microtubulesare nucleated in the “microtubule-organizing center,” at thecenter of the cell, and then extended one piece at a time intothe cytoplasm. Growth proceeds in fits and starts in a processknown as “dynamic instability.” Tubules grow slowly andsteadily, but are punctuated by periods of rapid disassembly,when large regions peel away from the ends. A small portionof the microtubule may break up, or the fragmentation mayextend all the way back to the start, completely destroyingthe tubule. In special cases, such as the microtubules thatsupport the long axons and dendrites of nerve cells, auxiliaryproteins stabilize the microtubule for longer periods of time.But the dynamic structure of microtubules is essential fortheir everyday function in transport and cell division.

Essential functions make excellent targets for toxinsand for cancer chemotherapy. The central role of micro-tubules in cell division makes them particularly attractive.Many organisms have developed toxins that block thedynamic instability of microtubules, thus blocking the abil-ity of the cell to divide. Two types of natural plant toxinsare widely used in medicine. They have the identical resultof blocking division, but achieve it in exactly oppositeways. The vinca alkaloids, such as vinblastine, vincristine,and vinorelbine, bind to the end of growing microtubules,blocking the addi-tion of more tubulindimers. The tubulecannot grow, but itcan still disassem-ble, so the micro-tubules ultimatelybreak down intonothing. Alterna-tively, the taxanes,

The Molecular Perspective: Microtubules and the Taxanes

DAVID S. GOODSELL

The Oncologist 2000;5:345-346 www.TheOncologist.com

Correspondence: David S. Goodsell, Ph.D., The Scripps Research Institute, Department of Molecular Biology, 10550North Torrey Pines Road, La Jolla, California 92037, USA. Telephone: 858-784-2839; Fax: 858-784-2860; e-mail:goodsell@scripps.edu WorldWideWeb: http://www.scripps.edu/pub/goodsell ©AlphaMed Press 1083-7159/2000/$5.00/0

Fundamentals of Cancer Medicine

TheOncologist

Figure 1. Microtubulestructure. Microtu-bules are composed oftwo similar proteins:alpha-tubulin (in blue)and beta-tubulin (inpink). Heterodimers ofone alpha and one betasubunit assemble into asturdy cylindrical tube.Paclitaxel (in green) binds to beta-tubulin on the inner surface, sta-bilizing the microtubule and blocking the normal dynamics of assem-bly and disassembly. Atomic coordinates were taken from entry 1tubat the Protein Data Bank.

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346 The Molecular Perspective: Simian Virus 40

such as paclitaxel and docetaxel, stabilize microtubules,blocking the disassembly process. When treated with tax-anes, cells are choked with large numbers of spurious astersforming throughout the cytoplasm.

Both the vinca alkaloids and the taxanes are large mol-ecules with complex chemistry. The multidrug transporter(discussed in this column in The Oncologist 1999;4:428-429) is designed with these types of molecule in mind, sodrug resistance can be a problem. Cancer cells overproducethe transporter and pump all of the drug outside, protectingtheir delicate flower of cell division from harm.

ADDITIONAL READING:

Amos LA, Lowe J. How Taxol stabilises microtubule structure.Chem Biol 1999;6:R65-R69.

Desai A, Mitchison TJ. Microtubule polymerization dynamics.Annu Rev Cell Biol 1997;13:83-117.

Mandelkow EM, Mandelkow E, Milligan RA. Microtubuledynamics and microtubule caps: a time-resolved cryo-elec-tron microscopy study. J Cell Biol 1991;114:977-991.

Nogales E, Whittaker M, Milligan RA et al. High-resolutionmodel of the microtubule. Cell 1999;96:79-88.

Figure 2. Microtubule dynamics. The assembly and disassembly of tubulin heterodimers (shown here as dumbbell-shaped molecules) intomicrotubules are controlled by GTP (guanosine triphosphate) molecules bound to the beta-tubulin subunits (individual GTP moleculesare not shown in this illustration). A tubulin heterodimer with GTP bound will add to a growing microtubule. Then, over time, the GTPis cleaved to form GDP, making the tubulin dimer less stable within the microtubule. But, as long as there is a cap of tubulin with GTPat the growing end, the microtubule as a whole will remain stable. But, if the GTP is cleaved at the end, a catastrophic disassembly willoccur, as shown at the left. At any given time, about half of the tubulin in a cell will be assembled into microtubules.

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David S. Goodsell: A Macrophage Engulfing a Bacterium

http://mgl.scripps.edu/people/goodsell/

The Macrophage

The Bacterium

Nucleus

Microtubule (Blue)

Actin filaments (Blue)

Blood

Ribosomes (Pink)

Nuclear pore (Green)

globulin G), was found to be conformation-specific. It did not recognize denatured tubu-lin by immunoblotting and seemed not to bindto native nonpolymerized tubulin. However,hMB11 cosedimented specifically with microtu-bules polymerized in the presence of guanylyl5′-(b,g-methylenediphosphonate) (GMPCPP), anonhydrolyzable GTP analog, and not with con-trol microtubules assembled in the presence ofGTP (Fig. 1A). In this experiment, low concen-trations of taxol (0.1 to 1 mM) were used to pre-vent depolymerization of control microtubules.When a higher concentration of taxol was used,hMB11 bound to both control and GMPCPP

microtubules (Fig. 1, B and C),which suggeststhat it recognized a conformation and not thenucleotide itself.

We then used hMB11 to stain by immu-nofluorescence a mixture of microtubules po-lymerized from pure tubulin in the presence ofGTP or GMPCPP (Fig. 1D). Under these con-ditions, hMB11 stained only GMPCPP micro-tubules [representing 68.6 T 17.3% (SD) ofMB11-positive microtubules] and not controlmicrotubules (1.8 T 0.9%). The remaining 29.7 T16.6% were bundles of both GMPCPP andcontrol microtubules. Despite varying experi-mental conditions, not all GMPCPP-containing

microtubules were stained by MB11, which sug-gests that some microtubules possessed confor-mational defects under these conditions.

Detection of tubulin in GTP conformationin cellular microtubules. We next used hMB11to localize GTP-tubulin in cellular microtu-bules by immunofluorescence. Because of itsconformational binding, hMB11 staining wasvery sensitive to structural alterations occurringafter fixation (10). It was best to use unfixedcells permeabilized in the presence of glycer-ol and/or low taxol concentration to preventmicrotubule depolymerization. In three repre-sentative cell lines (HeLa, Ptk2, and MDA-

Fig. 4. A GTP-remnant model for microtubule dynamic instability. (A)Model for microtubule dynamics showing GTP-tubulin (red) in a GTP capduring polymerization (P) and in inner microtubule regions. Upon caploss, the probability of catastrophe (C) increases and the microtubuledepolymerizes (D) until its end reaches a GTP-tubulin remnant. A GTP endis restored and the probability of microtubule polymerization increases,allowing its rescue (R). (B) Ptk2 cells stably expressing GFP-tubulin wereimaged at the indicated times. Rescue events (colored arrows) and the tipof a growing microtubule (arrowhead) are indicated. After cytosol extrac-tion, cells were stained with hMB11 (red) and imaged again, often show-ing GTP-tubulin remnants at rescue locations. Scale bar, 10 mm. The two

kymographs show the dynamics of the microtubules highlighted in redand yellow (top) aligned with hMB11 staining (bottom). Note the goodcoincidence of rescue position and GTP remnants. (C) Quantification ofexperiments done as in (A), showing the proportion of polymerizingmicrotubules stained by hMB11 at their plus ends and the proportions ofGTP-tubulin remnants that colocalized with rescue locations in Ptk2 cells(means T SEM). The proportion that would be expected in stochastic con-ditions is shown for reference at the right (Monte Carlo simulation, tableS1) (9). The table shows that the rescue frequency varies with the dis-tribution of GTP-tubulin remnants (means T SEM, comparison of Ptk2 andRPE1 cells) (9) (table S1).

www.sciencemag.org SCIENCE VOL 322 28 NOVEMBER 2008 1355

RESEARCH ARTICLES

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Microtubule Associated Proteins (MAPs):

Fig 20-12 Sf9 insect cells expressing either MAP2 (left) orTau (right

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Tau (green staining) is confined to the axon of ahippocampal neuron (long branched structure onthe left). whereas MAP2 staining (orange; isconfined to the cell body and to dendrites.

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Although this review will focus on the mechanigm ofplus-end tracLing and the regulation of plus-enddynamics, it is important to note that there has aleobeen significant recent progress in identifuing *TIP-interacting partners and in defining potential mechanismsofplus-end linkage to other cellulai structures [15 -22,88].These studies on attachment highlight the important roleof + TIPs in coordinating actin and microtubule-dependentprocesses. lhey also reveal new interactions important forcell division (euch as the interaction between microtubulesand the neck separating mother and daughter celle inbudding yeast) [23-25]. We refer readere to the citedpapers and to several excellent recent ieviews for furtherinformation [26-291.

Mechani3ms tor plus-ond trackingTreadmillingPlus-end tracking was first discovered by live-cellimaging of a fusion between the microtubule-associatedprotein (MAP) CLIP-170 and the green-fluorescentprotein (GFP) [30]. CLIP-170 was originally identifiedas a nucleotide-sonsitive microtubule-binding proteinin HeLa celle and wae subsequently shown to linkmicrotubules to endocytic vesicles in oitro 134,351. Atechnique called fluorescent speckle microscopy (FSM) [311demonstrated that the plus-end tracking of CLIP-170 ismediated by a treadmilling mechanism (Fig. 2). CLIP-170molecules appear to be added to the plus-ends of gropingmicrotubules, but shortly thereafter, these moleculesdissociate behind the region of new growth. This paradeof proteins coming on and then falling off the microtubuleend creates an optical illusion: although individualCLIP-170 molecules are stationary, the population ofCLIP-170 molecules appears ta surfon the growing ends ofmicrotubules as they rocket through t,Le cytoplasm.

Although CLIP-170 was recently found to have a numberofbinding partners, we suspect that plus-end tracking is anintrinsic property of the protein, based on the followingobservations t18,32-341. All CLIP-170 family members(CLIPs) tesied so far plus-end track [30,36-38]. TheCLIP prct€ins contain one or more consewed microtubule-binding domains, called the CAP-Gly domain at theirN-termini, The structure of the CAP-Gly domain wasrecently solved and shown to have a unique fold containingthree p sheets [39]. The CAP-Gly domain is followed by anc-helical domain and then in some CLIPs by a C-terminal'cargo-binding domain' containing one or more signaturezinc-binding motifs (CCHC motifs) [40]. CLIP-170 forms along extended homodimer arranged in a parallel orien-tation [4U. Deletion analysis revealed that short CLIP-170fragments containing little more than t,Le CAP-Glydornains can plus-end track when introduced into celle[301. Additionally, the CAP-Gly domain is not only foundin CLIPs but also in the p150or""d subunit of dynactin(a complex tlnt regulates the motor cytoplasmic dynein),in somo tubulin folding factors and in one member of theLinesin motor superfamily [39]. p150ar"'d was alsor€ceDtly shown to be a genuine +TIP by livo-coll imaging,and again a small fragment containing the CAP-Glydomain could plus-end track when introduced into cells[42J. Perhaps the CAP-Gly domain represente the bona

Fle.2. Thetr.admilling mechanish ior plus-6nd trackin€. Thtfigur. illusrrar.tthegrowth of 6 singlo microtubulo ov$ tim., +TlPs.,o 6dded wilh now growrh,

rsmain sbrion6ry wh6n bound {indic6r.d by tht v€rticsl d$hsd lin$) and rhonstochosrically dissoci6t6,

fide module for direct plus-end tracking; ho\c eve\ in oitroreconstitution will be needed to definitively rule out anintermediary between the CAP-Gly domain and thepolymerizing microtubule end.

Although treadmilling is well described, the under-Iying mechsnisms remain poorly understood. In prin-eiple, treadmilling could reeult from a higher afrnity of+TIP8 for the microtubule end .elative to the micro-tubule wall. fron a faster dissociation (release) fromthe microtubule wall than from the end, or both(Fig.3). The most favored mechanism for selectiverelease from the Eicrotubule wall is phosphorylation,which ie known to inhibit the binding of CLIP-170 andpl.1oct"'d tn microtubules [42,43]. Propoeed mechan-isms for higher-affinity plus-end binding include bind-ing to the GTP cap and recognition of a uniquestructural feature of the growing plue-end [441. Thefact that all +TIP8 can bind along the length ofmicrotubules when overexpreeaed highlights the deli-cate balance between binding and release required forselective plus-eud accumulation.

Tr€admilling as a mechanism fot plus-end tracking:ovidence for phoEphoryl ation-dependent selectivereleaac from the micrctubule wallFor CLIP-I?O, this delicate balance appears to be achievedby tlle combination of co-assembly of CLIP-170 with

tL (b)